A method of discriminating animal and plant individuals on the basis of a difference in DNA sequence is internationally important from the standpoint of clarifying the genetic background of test individuals and protecting the rights of breeders and fosterers dealing with the same. As a method of individual discrimination on the basis of a difference in DNA sequence is used a PCR (polymerase chain reaction) method. In order to perform individual discrimination by the PCR method, a base sequence of a primer (short DNA fragments, indispensable for the PCR) is an important factor for the success of the discrimination.
Various primers prepared on the basis of known or optional base sequences have been developed, and methods of individual discrimination by using these known or optional base sequences as a primer for the PCR have been proposed (Skolnick, M. H. and R. B. Wallace, Genomics 2: 273-279 (1988); Williams, J. G. K. et al., Nucleic Acid Res. 18: 6531-6535 (1990)); however, according to conventional methods, it has been often difficult to clearly ascertain different DNA fragments exhibiting polymorphism among test individuals and improvements thereof have been demanded intensely.
In addition, as a method of discriminating test individuals on the basis of a difference in DNA fragments can be mentioned methods proposed in RFLP (Restriction Fragments Length Polymorphysms) (Tangsley, S. D. et al., Biotechnology 7: 257-264 (1989)), the official gazette of Japanese Laid-Open Patent Publication No. 62-500423 (1987) and the official gazette of Japanese Laid-Open Patent Publication No. 6-504427 (1994).
It is guessed that base sequences having a symmetrical structure of a mirror image are scattered among DNAs of organisms. That is, for example, it has been thought generally that the phylogenetic differentiation of plant species occurred along with the change of their genes (DNAs) in the process of evolution of them. It can be guessed by comparing the sequences of genes (DNAs) of different plant species with each other that the DNAs were recombined through domains having common base sequences in the phylogenetic differentiation. The present inventor has engaged in studies about the structural change of DNAs of plant species using rice plant as test materials and has found that when the recombination of DNAs (homologous recombination) is caused through a domain having a common base sequence, the recombinant DNA with a base sequence having a symmetrical structure of a mirror image (mirror image structure) in some cases (Kawata, M. et al., Theor. Appl. Genet. 90: 364-371 (1995)). According to the above, it can be guessed that DNAs of organisms including rice plant, corn and the like contain a base sequence having a symmetrical structure of the mirror image as traces of the recombinations of DNAs caused in the process of evolution of them.
Under these circumstances, taking the above prior arts into consideration, the present inventor has engaged in assiduous studies with a view to developing a primer useful for a method of individual discrimination by the PCR method, and has investigated the efficiency of a base sequence with a symmetrical structure of such a mirror image as a primer, and as a result has found that discrimination of test individuals can be performed efficiently by using a base sequence with said mirror image structure as a primer for the PCR. That is, the present inventor has found that DNA fragments (MI primer) having a symmetrical base sequence with a mirror image structure found by the present inventor enable DNA fragments exhibiting polymorphism among test individuals to be observed more clearly as compared with a primer prepared on the basis of a known or optional base sequence, and that hence the use of the MI primer as a primer for the PCR is effective for individual discrimination, which has led to the accomplishment of the present invention.